Lipopolysaccharide (LPS) extracted from eight strains of Yersinia pestis, which had been cultured at 28 or 37°C, reacted equally well, in Western blots, with four monoclonal antibodies generated against the LPS from a single strain of Y. pestis cultured at 28°C. LPS was extracted and purified from Y. pestis strain GB, which had been cultured at 28°C. When the LPS was analysed by SDS-PAGE and MALDI-TOF mass spectrometry it was found to be devoid of an O-antigen. The LPS possessed activity of 2.7 endotoxin units/ng in the Limulus amoebocyte lysate assay. The LPS stimulated the production of TNFα and IL-6 from mouse macrophages, but was less active in these assays than LPS isolated fromEscherichia coli strain 0111. Y. pestis LPS, either alone or with cholera toxin B subunit, was used to immunize mice. Either immunization schedule resulted in the development of an antibody response to LPS. However, this response did not provide protection against 100 MLD of Y. pestis strain GB.